Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 61
DISCUSSION
DISCUSSION
61
Dr. Reinhold Benesch: I would like to draw attention to some work which
we have done which had as its aim the de nova introduction of an -SH group
into proteins through a peptide bond. Partly we had in mind the application
to x-ray work since the -SH groups introduced de novo in this way could be
transformed into mercury derivatives and the protein examined in this form.
The compounds which we selected for this purpose are homocysteine thio-
lactones, which would react with protein amino groups according to the fol-
lowing scheme ~ Benesch, R. and Benesch, R. E.: I. Am. Chem. Soc. 78: 1597,
1956):
S
CH.,
~ H.,
CH:3-CO-NH-CH-CO
+ PrNH~ ~
SH
CH
CH.,
ClI3-CO-NH-CH-CO-NH-Pr
Dr. David B. Smith: Regarding the number of subunits in hemoglobin, I
would like to bring to your attention some results from our laboratoryi~~~3 on
horse globin. Horse globin at pH 2 and ionic strength 0.05 separates into a
material whose weight-average molecular weight is 21-22,000 and whose
number-average molecular weight is 17,000. These results are interpreted
a. indicating four subunits with partial aggregation to give the higher weight-
average molecular weight. Molecular weights were measured by osmometry,
light scattering and sedimentation using Archibald's method.
Incidentally, the effect of pH 2 and ionic strength 0.05 on sedimentation
was checked in two ways. The sedimentation rate of ribonuclease in this
medium was the same as at neutrality. The molecular weight of lysozyme by
Archibald's method was about 14,000 in agreement with results obtained at
neutrality and higher ionic strength.
Under conditions where the molecular weight of globin has its minimum
value, that is pH 2 and ionic strength 0.05, the electrophoretic pattern is at
its simplest and shows two components. Extrapolation to allow for the dis-
torting effect of the extreme conditions on the relative areas of the peaks
indicates that the two components are present in equal amounts. We obtained
small amounts of each component from the ends of the electrophoresis appa-
ratus and investigated their properties separately.
The faster-moving component at pH 2 and ionic strength 0.05 had a weight-
average molecular weight of about 2S,000 and a number-average value of
about 17,000. Any increase in pH or ionic strength resulted in association.
The slower-moving component had a ~veight-average molecular weight of
about 17,000 and alterations in the medium had no effect on this value. The
OCR for page 62
62
PART I. STRUCTURE OF HEMOGLOBIN
behavior of unfractionated globin is in some respects intermediate between
that of these two components.
We have made some investigations by Edman's methods on the amino acid
sequence at the N-terminal ends of the separated components. Both compo-
nents, of course, have N-terminal valine. The second amino acid residue of
the faster-moving component is glutamic acid. The slower component has
principally leucine in the second position; slight contamination with glutamic
acid is ascribed to the difficulty of obtaining the slow component free from the
faster in the descending limb of the electrophoresis cell.
In conclusion, it appears that horse globin can be readily split into four
subunits, all of similar molecular weight and divided equally between two
types.
REFEREN CES
1. Reichmann, M. E.. and Colvin, J. R.: The number of subunits in the molecule of
horse hemoglobin, Can. J. Chem. 34: 411, 1956.
2. Haug, A., and Smith, D. B.: Separation, molecular weight and interactions of
horse globin components, Can. l. Chem., 35: 945, 1957.
3. Smith, D. B., Haug, A., and Wilson, S.: Physical and chemical studies on horse
globin components, Federation Proceedings 16: 766, 1957.
Dr. V. M. Ingram: Have you any information on human globin?
Dr. D. B. Smith: No.
Dr. b~al~er Hughes: I would like to report an observation which may be
important relative to heme-heme interaction. In searching for gentle methods
or removing heme from hemoglobin, I observed that approximately half of
the heme may be extracted from precipitated carbonmonoxy hemoglobin by
acetone containing small amounts of pyridine and water. The resulting product
appears very "native." It shows two peaks in the ultracentrifuge suggesting
partial dissociation into 34,000 M.W. units. I have not been able to remove
the remaining heme except by more rigorous conditions with concomitant de-
naturation. Myoglobin under these conditions releases no heme. If all of the
hemes are equivalent in hemoglobin, this finding must also be interpreted
through heme interaction, here of a negative (repulsive) nature. Lewist has
published a similar finding in the acid denaturation of carbonmonoxy hemo-
globin. However, he found the removal of only the first heme to be easier than
the rest.
REFEREE CE
1. Lewis, U. J.: The acid cleavage of hemoglobin, J. Biol. Chem. 206: 109, 1954.
Dr. M. T. Perutz: May I make a short point? I should like to remind you
of a result of Kendrew and Parrishi which has some bearing on the crevice
theory of iron attached in myoglobin. They prepared the 1-methyl and 4-
methyl imidazole derivatives of myoglobin, which thus have large groups
attached to the iron atom. They crystallized those compounds and took x-ray
OCR for page 63
DISCUSSION
63
pictures. In taco species of myoglobin the imidazole group produced no change
in the unit cell dimensions of the crystal. If there were the kind of crevice
where the molecule is forced apart, as it were, through the insertion of a
group like propyl isocyanide, then this ought to have the effect of making
the molecule somewhat bigger and enlarging the unit cell. This, as I say, was
not observed. In a third species crystallization in the usual crystal form was
inhibited and replaced by another. Kendrew and Parrish conclude that the
heme is most likely to be on the surface of the myoglobin molecule, the imi-
dazole group finding space in the interstices between neighboring molecules
in the crystal lattice.
REFEREN CE
1. Kendrew, J. C. and Parrish, R. G.: Imidazole complexes of myoglobin and the
position of the haem group, Nature (Lond.) 175: 206, 1955.
Dr. Davidson: Dr. Perutz, would you not expect something like PC~B to
change the size of a hemoglobin molecule?
Dr. Edsall: You mean whether there is or is not a crevice so that merely
tacking on a group as large as that to a hemoglobin will alter its dimension?
Dr. Perufs: In the picture vou saw here the distance between the -SH
groups was 30 Angstroms. This picture does not tell you whether the -SlI
groups are on the surface or within the molecule. However, further results
have now been obtained by Dr. David Green at the Royal Institution in
London, which seem to show that the -SH group is about 7 Angstroms in-
side the external boundary of the molecule. In other words, there must be
a sort of crevice or canal where the -SH group is located, so that the PCMB
does not make the molecule any bigger.
I should like to mention a paper published by Drs. David Ingram. Gibson
and myselt,l concerning the orientation of the neme groups. We measured
the paramagnetic resonance or electron spin resonance, as it is sometimes
called, of the iron atoms in single crystals of horse me/hemoglobin. We got
a very beautiful and sharp anisotropic effect with the help of which it was
possible to determine the angular orientation of the heme groups with a very
high accuracy indeed.
The accompanying figure ~ shows the hemoglobin molecule, egg shaped,
55 Angstroms wide, 55 Angstroms thick and 70 Angstroms long, lying on
an axis of dyed symmetry (the lo-axis). The heme groups are arranged in
two pairs, related by the dyed axis. One pair of heme normals lie in the
a,b-plane of the crystal, while the other pair is tilted by 13° above and below
that plane. I should like to stress that this result tells nothing about the
position of the heme groups. As figure 2 shows, they can be anywhere you
like, except that they must be related in pairs by the two-fold axis of symmetry.
In order to find their position, we made the para-iodo-nitroso-benzene com-
plex of hemoglobin, hoping that the iodines would label the iron atoms and
OCR for page 64
64 PART I. STRUCTURE OF HEMOGLOBIN
/c
g ~~ ~'
~ /
~,'~
dead
_ ~emoglo~
/~N ~
·Fe ~
F[G. 2. Three of the many possible
arrangements of the four heme groups
in the hemoglobin molecule, repre-
sented diagrammatically as a spheroid.
In each pair of drawings the left-
hand spheroid shows the molecule in
projection normal to the dyed axis,
and the right^hand one shows the
same arrangement seen along the dyed
axis. The small black points in ( c)
represent the positions of the -SH
groups deduced from the x-ray anal-
ysis. (From Nature 178: 908, 1956.)
FIG. 1.—Perspective drawing of the
orientation of the heme groups with
respect to the crystal axes and the
hemoglobin molecule. ( a ) and ( b )
show the two pairs of heme groups
related to the dyed axis; (c) shows
the external shape of the molecule, as
determined by }3ragg and Perutz,0
drawn on a much smaller scale than
the heme groups. (From Nature 178:
907, 1956.)
a) \ a_ IT'
Ah /~ ': ~ a
a
a
' 1
~ /'' (C) in\—i~
~ THAI
OCR for page 65
DISCUSSION
help us to find their positions in an electron density map. Unfortunately,
the results obtained so far have been inconclusive.
REFERENCE
65
1. Ingram, D. J. E., Gibson, J. F., and Perutz, M. F.: Electron spin resonance in
myoglobin and haemoglobin. Orientation of the four haem groups in haemo-
globin, Nature (Lond.) 178: 905, 1956.
Representative terms from entire chapter:
ionic strength
